Abstract

Background and Objectives: We sought to incorporate novel agents into multimodality therapy for poor-prognosis human malignancies, specifically neuroblastoma and glioma. Neuroblastoma is the most common extracranial tumor of childhood and high-risk patients with evidence of metastases have an overall survival rate of less than 40% despite intensive multi-modality treatment and therefore new treatment strategies are urgently needed. For neuroblastoma, targeted radiotherapy with 131I-metaiodobenzylguanidine (MIBG) is a promising new treatment approach with 30% response rates in refractory disease. MIBG, which is a norepinephrine analogue, is taken up by the norepinephrine transporter (NET) that is abundantly expressed on neuroblastoma cells. We have focused on combining 131I-MIBG with novel drugs, likely to function as radiosensitizing agents. Specifically, we investigated the addition investigated the radiosensitizing effect of vorinostat (suberoylanilide hydroxamic acid) and radiation in the treatment of metastatic neuroblastoma in vitro and in vivo and evaluated potential mechanisms underlying these interactions.

Due to their molecular heterogeneity and infiltrative nature, glioblastomas (GBM) have been notoriously difficult to treat with traditional and experimental therapeutics. Similarly, neuroblastoma the most common extracranial tumor of childhood, often presents with high-risk disease, and such patients have an overall survival rate of less than 40% despite intensive multi-modality treatment. Thus, for both glioblastoma and neuroblastoma new treatment strategies are urgently needed. To overcome the hurdles of resistance to treatment, many have suggested the use of targeted agents in combination with conventional therapy and we have sought a rational approach to the choice of targeted agent for each of these malignancies. Since the PI3K/mTOR pathway is known to play a critical role in glioma pathogenesis, we chose to target this pathway using an orally bioavailable PI3K/mTOR inhibitor (XL765). We evaluated the preclinical potential of XL765 in the treatment of gliomas, in in vitro and in vivo models, as a single agent and in combination with radiation, temozolomide, and erlotinib.

Methods: For the study of neuroblastoma, various neuroblastoma cell lines were exposed to radiation, vorinostat, and combinations thereof. Response to therapy in vitro was assessed using an ATP-based proliferation assay and clonogenic survival curves. To establish the effects of vorinostat and radiation in vivo, a metastatic neuroblastoma model was established by tail vein injection of NB1691luc cells into athymic mice (a generous gift from Dr. Dickson, St. Jude Children's Research Hospital). Mice developed widely metastatic tumor that was visible by bioluminesence 21 days post-injection in 100% of animals. Mice were treated with vorinostat 150 mg/kg intraperitoneal (IP), radiation, or combinations thereof. Each treatment group contained 10 mice. Since in all tumor-bearing mice metastatic neuroblastoma was detected in all parts of the body except the head, 1 Gy of radiation was administered to the entire animal's body while shielding the head. Radiation was performed 1 hour after IP administration of either vorinostat or DMSO as a control. Phosphorylated H2AX after treatment with vorinostat and radiation was determined by flow cytometry. Western blot analysis was performed to detect variations in expression levels of DNA repair enzymes Rad51 and Ku-86.

For the study of glioblastoma, we have obtained a series of in vivo serially passaged human GBM xenografts termed GBM6 (EGFR VIII amplified, PTEN wt), GBM8 (EGFR wt amplified, PTEN null), GBM12 (EGFR wt amplified, PTEN wt), GBM 39 (EGFR vIII amplified, PTEN wt), and GBM GS-2 (EGFR wt, PTEN null) that are more genetically stable than GBM cell lines in culture. Using XL765 in vitro we have evaluated downstream molecular changes by Western blot and cytotoxicity by colorimetric ATP-based assay. Based on these results we have combined XL765 with erlotinib (ERL), temozolomide (TMZ) and radiation (XRT) using the same assays to test for combinatorial effects. Finally, we injected xenograft GBM39 (EGFR VIII amplified, PTEN wt) intracranially into nude mice and tested various combinations of the above agents. GBM39, altered to express luciferase, was used so that intracranial tumor burden could be monitored using optical imaging.

Results: For studies focusing on neuroblastoma, vorinostat resulted in dose-dependent decreased viability in all three independent neuroblastoma cell lines tested. Vorinostat had radiosensitizing effects in vitro in all three cell lines investigated. Animals treated with a combination of vorinostat and radiation showed decreased tumor volumes compared to single modality treatments (p(2) = 0.04). Phosphorylated H2AX was increased after treatment with vorinostat and radiation compared to radiation or drug alone. Expression levels of Ku-86 were reduced in neuroblastoma cells treated with vorinostat, whereas Rad51 expression did not change.

In the study of glioblastoma and the PI3K/mTOR inhibitor XL765, for all xenografts tested in vitro, XL765 inhibited the PI3K pathway in a concentration- and time-dependent manner and these molecular changes correlated with increasing cytotoxicity. Treatment of GBM6 with XL765 + TMZ + XRT resulted in combined cytotoxicity that was significantly greater than any monotherapy or dual therapy combinations (p = 0.002). Similarly, in GBM8 combination of XL765 + TMZ demonstrated increased cytotoxicity compared to either agent alone (p < 0.001). In GBM12 and GBM GS-2 the combination of XL765 + XRT was more cytotoxic than either therapy alone (both p < 0.005). Combination of XL765 with ERL showed supra-additive effects on cytotoxicity in all xenografts (p < 0.05). In vivo, XL765 administered by oral gavage showed a greater than six-fold decrease in tumor bio-luminescence compared to control (Mann-Whitney test p(2) < 0.0001). TMZ alone showed a ten-fold decrease in bio-luminescence, but combination with XL765 yielded a >60-fold reduction in bio-luminescence (p(2) < 0.0001). Combination of XL765 + ERL + TMZ resulted in regression of bio-luminescence below baseline levels (p(2) < 0.0001). Interestingly, erlotinib was remarkably effective in vivo against GBM 39 (p(2) = 0.0002 for erlotinib compared to control), a finding that is not surprising given the permissive genotype of this particular xenograft, containing both EGFR vIII amplification AND wild type PTEN. Also consistent with this genotype is the finding at inhibition of PI3K/mTOR did not add further to the cytotoxicity of erlotinib in this particular xenograft (p(2) = 0.81 for erlotinib+XL765 compared to erlotinib alone).

Conclusions: In the multimodality treatment of high-risk neuroblastoma, vorinostat has a radiosensitizing effect on neuroblastoma cells in vitro and in vivo. A possible mechanism of radiosensitizing is down-regulation of the DNA double strand repair enzyme Ku-86. These preclinical results have been translated into a phase 1 clinical trial of the combination of vorinostat and 131I-MIBG.

In the multimodality treatment of gliomas, the PI3K/mTOR inhibitor XL765 showed excellent single agent cytotoxicity and resulted in supra-additive anti-tumor activity when combined with temozolomide in the treatment of a xenograft with MGMT hypermethylation. A glioma with MGMT hypermethylation would be expected to be relatively sensitive to temozolomide and we are currently asking whether such cooperative anti-tumor activity will also be evident in gliomas without MGMT methylation. Conversely, XL765 did not add to the cytotoxicity of erlotinib, again, as predicted by genotype. Against the permissive genotype of both EGFR vIII amplification and wild type PTEN, erlotinib alone was exceedingly effective as a single agent and did not require inhibition of PI3K to enhance its anti-tumor activity. We predict that XL765 will cooperative with erlotinib in decreasing tumor growth in gliomas with NON-permissive genotypes such as those with PTEN mutations and we are currently testing this hypothesis in vivo.